Membrane fusion, a process critical for viral entry, is promoted by the paramyxovirus fusion (F) proteins. All F proteins contain a number of common features that play fundamental roles in fusion. However, significant variations exist between F proteins related to protein activation and promotion of membrane fusion, and many critical questions remain concerning the mechanism by which fusion is promoted by these important viral proteins. The long-term objective of our research is to understand the precise mechanism(s) of paramyxovirus F protein-promoted membrane fusion. Our overall hypothesis is that the function of domains critical for fusion promotion will be conserved in diverse F proteins, but that interactions in these domains will modulate triggering mechanisms. To address this important hypothesis we will pursue the following specific aims: 1.) We will build on our exciting preliminary data which demonstrates trimer formation of isolated Hendra F transmembrane (TM) domains to define the role of TM-TM interactions in glycoprotein folding and fusion. We will therefore delineate the residues critical for TM-TM interactions, and evaluate the role of this interaction in Hendra F folding, trafficking and fusion; analyze the effect of HRB or cytoplasmic-tail additions on stability of TM-TM interactions; compare TM-TM interactions for the PIV5 and HMPV F proteins to those observed for the Hendra F TM; and determine if TM domains from paramyxovirus attachment proteins interact with themselves or with the F protein TM domains; 2.) We will define the role of low pH and endocytosis in HMPV viral entry and delineate the role of electrostatic repulsion in the HRB linker region in HMPV F low pH-induced conformational changes and entry of recombinant viruses and; 3.) We will analyze the role of stabilizing interactions between HRA and a conserved region of F2, which our studies of PIV5 F indicate can play a role in triggering, by defining key interactions in this domain in PIV5 F, assessing the stage of fusion which is affected, analyzing the effect of mutations in this region on viral entry, and analyzing the effect of mutations to this region in other F proteins. Accomplishing these goals will provide crucial information on the regions that control the molecular events involved in the F protein-promoted membrane fusion process, and potentially identify new targets for antiviral therapy. PUBLIC HEALTH RELEVANCE The paramyxovirus family contains both established human pathogens, such as measles virus and respiratory syncytial virus, and newly emerged human pathogens, including the highly pathogenic Hendra and Nipah viruses and the recently identified human metapneumovirus (HMPV). A detailed understanding of the fusion mechanism could lead to development of new antiviral reagents.